Structural Analysis Provides Mechanistic Insight into Nicotine Oxidoreductase from Pseudomonas putida

Abstract

The first structure of nicotine oxidoreductase (NicA2) was determined by X-ray crystallography. Pseudomonas putida has evolved nicotine-degrading activity to provide a source of carbon and nitrogen. The structure establishes NicA2 as a member of the monoamine oxidase family. Residues 1-50 are disordered and may play a role in localization. The nicotine-binding site proximal to the isoalloxazine ring of flavin shows an unusual composition of the classical aromatic cage (W427 and N462). The active site architecture is consistent with the proposed binding of the deprotonated form of the substrate and the flavin-dependent oxidation of the pyrrolidone C-N bond followed by nonenzymatic hydrolysis.

Figure 1

Ribbon diagram of NicA2 colored by secondary structure (helices in green, sheets in purple and loops in cyan). FD represents the FAD-binding domain; SD-I and SD-II represent the substrate-binding subdomains. The FAD cofactor is depicted as yellow sticks.

Figure 2

Stereoview of the noncovalently bound FAD co-factor. Unbiased electron density corresponding to FAD before refinement with model is shown as an omit (Fo-Fc) map at a 2σ contour level. Residues homologous to the aromatic cage are depicted as green sticks.

Scheme 1.

Aminoketone pathway of L-nicotine degrada-tion in P. putida via NicA2 and A. nicotinovorans via 6HLNO.